Anti-inflammatory Activity of Sting Protein from Apis mellifera

Int. J. Life. Sci. Scienti. Res., 3(2): 914-919 MARCH- 2017
http://ijlssr.com Copyright © 2015-2017 International Journal of Life-Sciences Scientific Research Page 914
Anti-inflammatory Activity of Sting Protein from
Apis mellifera
Sumesh Shah1
, Amit Gupta2, 3*
, Shweta P Karne1
, Sanjay Kamble1
, Bharat Shinde1,2, 3
1
Department of Microbiology, Vidya Pratishthan’s, Arts, Science and Commerce College, Baramati, Maharashtra, India
2
Department of Zoology, Vidya Pratishthan’s, Arts, Science and Commerce College, Baramati, Maharashtra, India
3
Department of Immunology and Virology, Vidya Pratishthan’s School of Biotechnology (VSBT, Research Centre
affiliated to Savitribai Phule Pune University), Baramati, Maharashtra, India
*
Address for Correspondence: Dr. Amit Gupta, Assistant Professor/Senior Scientist, Department of Biotechnology,
Vidya Pratishthan’s, Arts, Science and Commerce College, Baramati, Maharashtra, India
Received: 13 January 2017/Revised: 31 January 2017/Accepted: 10 February 2017
ABSTRACT- The objective of our study is to determine its anti-inflammatory potential of protein extracted from the
stings of honey bee (Apis mellifera). In this study, protein extracted from the stings of Apis mellifera using Tris HCl/ice
cold acetone and determined through Nano drop method and then determined its Da protein using SDS-PAGE. In
addition, indirect ELISA was performed using rubella vaccine as coating antigen and determined its antibody titre using
variable concentration of sting protein (15.62-250 µg) and also determined its activity on human whole blood for
determining total cellular content and proliferation against rubella vaccine antigen. The results showed that protein from
stings of Apis mellifera showed drastic declined in antibody titre at higher doses but there is slightly enhancement in
antibody titre, total cellular content and proliferations at lower concentration as compared to control and rubella vaccine
(standard).Overall, this study suggest that stings protein of Apis mellifera showed anti-inflammatory potential against
rubella vaccine antigen.
Key-words- Anti-inflammatory, Apis mellifera, Stings, Nanodrop, ELISA
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INTRODUCTION
Apitherapy is considered to be one of the most important
practiced in many cultures whereas bee venom therapy is
used in the form of live bee stings in order to treat various
diseases e.g. arthritis, multiple sclerosis, diseases of the
central and peripheral nervous system, heart and blood
system, skin diseases and other diseases [1-3]. In general,
honeybee venom is generally produced by two glands
which is associated with sting apparatus of worker and
queen bees. The production of bee venom increased during
first two weeks of life and reached up to maximum when
they become involved in hive defence and foraging [1,4-5].
In contrast, honey bee venom showed complex variety of
numerous peptides including proteins that showed strong
neurotoxic and immunogenic effects.
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DOI: 10.21276/ijlssr.2017.3.2.7
As per the literature, most active component that is present
in major amount of bee venom i.e, mellitin which showed
antimicrobial activity [6-7].
Among many species of insects belongs to the Phylum
Arthropoda, only few of them have the ability to defend
themselves with a sting or venom injection during stinging
[3, 8]. Most of these insects that can sting which represents
the members of the order Hymenoptera (includes ants,
wasps and bees). Normally, stings are always present at the
abdominal end of Apis mellifera and pain inflicted by Apis
mellifera, defending its colony, is not caused by a bite, as is
frequently said, but by a sting [9]. In the present study, we
focused on protein extracted from the stings of Apis
mellifera and determined its immunological studies against
specific protein antigen i.e. rubella vaccine.
MATERIALS AND METHODS
Collection of samples
Twenty five Apis mellifera (females, worker) were
collected in the month of January, 2017 from Apairy,
VSBT bee hive. Apis mellifera, workers were pressed
manually using forceps and placed on glass slide. Then
Apis mellifera begin to release viscous liquid from stings
and were collected in glass slide and kept in a sterile
condition in order to avoid contamination. Thereafter,
Research Article (Open access)

Int. J. Life. Sci. Scienti. Res., VOL 3, ISSUE 2
viscous liquid was dried at room temperature for estimating
protein content using Nano drop method.
Estimation of protein content
In this study, dried sample of viscous fluid from stings was
taken in eppendorf tube and then add extraction buffer (i.e.
20 mM Tris HCl) dissolved in PBS, pH 7.4. Incubate
sample along with extraction buffer for 5-8 minutes (room
temperature) and centrifuging(6000 rpm; 10 minutes at
4°C) it. Supernatant was collected and then add equal
volume of ice cold acetone. Incubate the samples for 10-15
minutes at room temperature and then centrifuged. Collect
the pellet and washed with PBS. Finally, protein
concentration was determined by using Nano drop method
[10].
SDS-PAGE
In this study, we used resolving (15 %) and stacking (4%)
gels for determining the sting protein bands of Apis
mellifera. For SDS-PAGE, using protein sample extracted
from stings of Apis mellifera was loaded into the wells
(voltage, 50 Volts was required) and run it for 5 h. After
loading the samples, separation of protein bands of sting
isolated from Apis mellifera through electrophoresis,
staining solution was required to stain the gel of sting
protein in order to make bands visible. Afterwards the gel
was placed in to a de-staining solution for 24 h on shaker
and was changed frequently until clear gel was obtained.
ELISA
For determination of antibody (IgG) titre against Rubella
vaccine (Serum institute of India Limited, India) using
variable doses of protein extracted from stings of Apis
mellifera. In this study, Elisa plates were coated overnight
with Rubella vaccine (1:500 dilutions, 100 µl) in high bind-
ing 96 well plate (Himedia, India). Wash the plate with
PBS and then add 1 % BSA (bovine serum albumin; 100
µl) in 96 well plate. Incubate the plate for 1 h at room
temperature and then again wash the plate with PBS.
Thereafter, add variable concentration of protein
(15.62 – 250 µg) and incubate it another 4 h at carbon
dioxide incubator. After incubation, wash again with PBS
and then add horse anti-serum (1:1000 dilution; 100 µl)
used as secondary antibody. Incubate the plate for another 1
h at carbon dioxide incubator. After incubation, trimethyl
benizidine (TMB, 100 µl) substrates were added and keep
it in dark for 15 minutes and then add stop solution (1N
H2SO4). The optical density was measured at 450 nm [11].
Total cellular content
Virally infected whole blood samples (n=5) of human were
collected from Mangal pathology lab, Baramati, District
Pune, Maharashtra. For determination of total cellular
content using humanwhole blood (100 µl) was taken in
each falcon tube along with rubella vaccine (1:1000
dilution; 10 µl) and variable concentration of protein
(15.62-250 µg). Incubate the blood samples containing
protein and rubella vaccine for 2 h at room temperature.
Thereafter, lysis (red cell lysis buffer), and washing these
samples once with PBS (pH 7.4). Finally, the samples were
again dissolved in PBS and centrifuged at 10,000 rpm
(4ºC, 10 minutes) and supernatant was collected for
estimating total cellular content using Nanodrop method
[12].
Proliferation assay
Studies were conducted in human whole blood samples
(n=5; 100 µl) and exposed to rubella vaccine (1:1000
dilution, 10 µl) along with variable concentration of protein
(15.62-250 µg). Incubate these samples related to protein in
96 well plate for 24 h incubation in carbon dioxide
incubator. After incubation, add MTT solution (5 mg/ml,
10 µl) and then incubate it for another 4 h at carbon dioxide
incubator. Finally, centrifuging the samples after
incubation, fresh formazan crystals were settled at the
bottom and these crystals were dissolved in dimethyl
sulphoxide (DMSO) in a final volume of 0.2 ml. The
optical density (OD) was measured at 570 nm [13].
Statistical analysis
The difference between control and variable doses of
protein extracted from viscous fluid of stings from Apis
melliferais determined through one way ANOVA test
(Bonferroni multiple comparison test).
RESULTS
Estimation of protein
Immunobiological studies of protein extracted from the
stings of Apis mellifera showed protein content (10 µl,
0.978 mg/ml) which is determined through Nano Drop as
shown in Fig. 1 and its protein in the form of Dalton (Da)
which is confirmed through SDS-PAGE and its range
between 2050 – 2810.

Fig. 1. Estimation of protein content from viscous fluid (Bee venom) of stings from Apis mellifera
ELISA
The results of these studies related to anti-rubella antibody titre using variable concentration of protein (15.62 - 250 µg) as
shown in Fig. 2. The results showed that protein declined in antibody production at higher doses as compared to rubella
vaccine control. In other words, protein from stings could be a potent anti-inflammatory agent.
Fig. 2. ELISA assay. Indirect ELISA was assayed using rubella vaccine as coating antigen using variable doses of
sting protein for determining antibody titre. Horse anti-serum used as secondary antibody. The difference between
control and variable doses of protein is determined through one way ANOVA test (Bonferroni multiple
comparison test)
*p< 0.05; **p< 0.01 and ***p< 0.001

Total cellular content
Immunobiological studies were conducted in order to estimate total cellular content in lysed human whole blood using
variable concentration of protein (15.62 – 250 µg) in presence of rubella vaccine as shown in Fig. 3. The results showed
that protein showed declined in total cellular content at higher doses as compared to rubella vaccine control.
Fig. 3. Estimation of total cellular content in human whole blood. Lysed human whole blood was cultured with
variable concentration of sting protein (melittin) in presence of rubella vaccine. After incubation, lysis and washing
(PBS, pH 7.4) the samples after centrifugation at speed (10000 rpm) for estimating total cellular content using
Nanodrop method
Proliferation assay
The effect of variable concentration of protein (15.62 – 250 µg) extracted from the stings of Apis mellifera stimulated
proliferative response in lysed human whole blood along with rubella vaccine as shown in Fig.4. The results showed that
there is dose dependent decline in proliferation as compared to rubella vaccine control. Overall, the data indicates that
protein from stings of Apis mellifera inhibits T cell proliferation.
Fig.4. Proliferation assay. Lysed human whole blood was cultured with variable concentration of sting protein in
presence of rubella vaccine. After incubation, centrifuge the samples and add MTT solution (5 mg/ml, 10 µl). Fresh
formazan crystals were appeared and settled at the bottom and then finally dissolved in dimethyl sulphoxide
(DMSO) in a final volume of 0.2 ml. The optical density (OD) was measured at 570 nm. The difference between
control and variable doses of protein is determined through one way ANOVA test (Bonferroni multiple
comparison test)
*p< 0.05; **p< 0.01, and ***p< 0.001

DISCUSSION
Inflammation (means tissue injury or complex cascade of
nonspecific events) is generally triggered by innate and
adaptive immune systems in order to maintain homeostasis.
The term inflammation is usually classified or described in
the form of acute and chronic inflammation. These two
types of inflammation are usually classified on the basis of
cell types that take part in the inflammatory response. One
of the major cause of acute inflammation i.e. microbial
infections, hypersensitivity reactions, chemicals etc. in
contrast, if some agent causing acute inflammation and is
not properly removed or treated, it may progress to the
chronic stage of inflammation. In other words, the
physiological response to tissue injury (infection, surgery,
radiation etc.) involves both local and systemic reactions.
Normally, inflammatory responses are responsible to
maintain homeostasis condition and allows for tissue
healing whereas chronic inflammation leads to multiple
organ dysfunctions. So, there is an intimate relationship
between the mechanism of inflammation and the immune
system response [14-15].
As per the literature, bee venom extracted from the stings
of Apis mellifera has been widely used medicinally in
Europe for the treatment of rheumatic diseases. During
ancient period, healers have practiced apitherapy with
respect to honeybee products that are used for various
curative purposes [2-6]. As per the literature, researchers
focused or explored the potential of bee venom extracted
from stings for treating a wide variety of conditions from
acute tendonitis to chronic back pain to rheumatoid arthritis
[1]. In the present study, we focused on protein extracted
from stings and determined its immunological studies
against specific protein antigen i.e. rubella vaccine.
In the present study, we determined its immunological
effect of protein from stings on rubella vaccine using
human whole blood and showed that protein at higher
doses showed anti-inflammatory effect against rubella
vaccine antigen. This vaccine is used as standard for these
studies for determining T cell activation or proliferation
using variable concentration of sting protein. As per the
literature, this protein i.e. melittin have the capability to
reduce inflammation rate at higher doses and also showed
anti-inflammatory effect at lower doses [16]. Similar types
of results were also observed and showed
anti-inflammatory effect. Lot of research work is done
related to melittin protein and showed several
anti-inflammatory mechanisms in different types of disease
models. Similarly, bee venom and its melittin with respect
to anti-arthritic mechanism which directly targets in order
to inactivate NF-κB through direct binding of p50 subunit
[17].
As per the literature, in vitro assays were already performed
and revealed the potential of melittin as an effective agent
for the prevention of various neurodegenerative diseases. In
addition, melittin also showed potent immunosuppressive
effect with respect to pro-inflammatory responses of BV2
microglia and claimed that melittin may have potential to
treat various neurodegenerative diseases which is
accompanied with microglial activation [18]. Lot of
research work related to melittin protein is already done
and considered as allergenic peptide (major constituent of
apitoxin) which is responsible for cell lysis including cell
death [19].
Studies were also conducted in order to determine its
proliferation assay and total cellular content on human
whole blood using rubella vaccine. The results showed that
sting protein showed drastic decline in rubella vaccine
proliferation and total cellular content at higher doses as
compared to control. In other words, sting protein showed
cytotoxic effect against rubella vaccine using human whole
blood in a dose- dependent manner. Overall, the data
showed that protein from stings showed anti-inflammatory
effect against rubella vaccine antigen.
CONCLUSION
In this study, our result showed that sting protein should be
possibly used for anti-inflammatory agents only if careful
provisions or supervision are taken in order to avoid
adverse effects. In our next study, we tried to determine or
explore or synthesized protein derivatives for developing
novel pharmaceutical agents. The future therapeutic
application of melittin on inflammatory disorders will
depend on new study protocols to validate the efficiency
and safety of sting protein.
AUTHORS CONTRIBUTION
This work was carried out in the collaboration between five
authors. Amit Gupta designed the study, wrote the protocol
and interpreted the data where SS anchored the field study,
gathered the initial data related to his M.Sc Microbiology
dissertation work under Amit Gupta guidance and
performed preliminary data analysis. Amit Gupta, Sumesh
Shah, Shweta P Karne, Sanjay Kamble, and Bharat Shinde
managed the literature searches whereas AG and SS
produced the initial draft. The final manuscript has been
read and approved by all authors.
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How to cite this article:
Shah S, Gupta A, Karne SP, Kamble S, Shinde B: Anti-inflammatory Activity of Sting Protein from Apis mellifera. Int. J.
Life. Sci. Scienti. Res., 2017; 3(2): 914-919. DOI:10.21276/ijlssr.2017.3.2.7
Source of Financial Support: Nil, Conflict of interest: Nil

Anti-inflammatory Activity of Sting Protein from Apis mellifera

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